Recovery and analysis of MeIQx-DNA adduct by the 32P-postlabeling method

268 lular RecA. They are ZA180 (ada), TN2185 (ada recA), TN2183 (ada lexA) and TN2183/pKY102 whose plasmid carries the recA ÷ gene. O6-MeGT activity in each crude cell extract was evidently different. Its activity was greatest in TN2185, 9 times higher than in TN2183/pKY102 which was the lowest. On the other hand, the mutation frequency induced by MNNG was lowest in TN2185 and highest in TN2183/pKY102. These results suggest the possibility that some interaction between RecA and O6-MeGT may exist and that the interaction may be implicated in MNNG mutagenesis.

61 Ochiai, M., M. Hirose, K. Wakabayashi, T. Sugimura and M. Nagao, Carcinogenesis Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104 (Japan)

Recovery and analysis of MelQx-DNA adduct by the 32p-postlabeling method Heterocyclic amines (HAs) are contained in cooked foods and most of them are carcinogenic to mice and rats. In HAs, 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MelQx) is abundant in cooked foods and showed strong carcinogenicity to rodents. Analysis of H A - D N A adduct levels in humans will give us important information to estimate the risks of HAs. We first analyzed M e l Q x - D N A adducts in rats using the 32p-postlabeling method. M e l Q x - D N A adducts were detected by the nuclease P1 method with the same efficiency as the standard method, but the detection limit of the former was much lower. Thus all experiments were carried out with the nuclease P1 method. The liver of a rat fed MelQx gave 3 major adducts. To characterize these adducts, nitro-MelQx was chemically converted to N-hydroxy-MelQx, and reacted with 4 different deoxynucleoside-3'monophosphates in the presence of acetic anhydride. Each MelQx-adducted nucleotide was postlabeled and analyzed by co-chromatography. The most abundant spot obtained from in vivo modified DNA was divided into at least 2 adducts: one derived from guanine, and the other sug-

gested to be derived from guanine a n d / o r adenine. The recovery of Mel Q x-D N A adducts by the nuclease P1 method was analyzed by i.p. dosing of 14C-MelQx. DNA was purified by CsCI equilibrium centrifugation. The DNA adduct level detected by the nuclease P1 method was found to be about 50% of that detected with 14C radioactivity. Based on the data of contents of MeIQx in foods, the 32p-postlabeling method, with some modifications, should be useful to detect MelQxDNA adducts in humans.

62 Ohe, T. 1,2, M. Tokuda ~, K. Nakamiti 1 and T. Abe 1, I Department of Hygiene, Kyoto Prefectural University of Medicine, Kyoto 602 and 2 Department of Hygiene, Kyoto Women's University, Kyoto 605 (Japan)

Genetic susceptibility to etoposide-induced chromosome aberration formation and sister-chromatid exchanges (SCEs) in cultured human lymphocytes We have studied antitumor drug-induced SCEs and chromosome aberration formation in cultured human lymphocytes. In the study reported here, we examined SCEs and chromosome aberration formation induced by etoposide on 3 human lymphocyte cultures from normal subjects. The mitotic index decreased abruptly at 10 - 6 M etoposide and the frequency of SCEs was dose-related; a marked increase was recorded at 5 )< 10 - 6 M etoposide in the 3 subjects. A dosedependent effect was also found for chromosome aberration formation at concentrations between 10 -1~ and 10 -6 M. The aberrations observed were primarily chromatid breaks, chromatid gaps and chromatid exchanges. There was different susceptibility to etoposide-induced chromosome aberration formation among the 3 normal subjects. We also examined the individual differences in etoposide-induced chromosome aberration formation in 15 human lymphocyte cultures (11 normal subjects and 4 cancer patients) in order to clarify the genetically controlled susceptibility to